Recombinant Putative antitoxin VapB17 (vapB17)

What is the biological function of VapB17 antitoxin in Mycobacterium tuberculosis?

VapB17 functions as the antitoxin component of the VapBC17 toxin-antitoxin system in M. tuberculosis. Based on characterization of similar VapB antitoxins, VapB17 likely serves multiple roles: neutralizing its cognate VapC17 toxin to prevent growth inhibition, regulating gene expression by binding to the promoter-operator region of the vapBC17 operon, and participating in stress adaptation mechanisms. TA systems in prokaryotes have been implicated in various biological processes including post-segregational killing, stress adaptation, phage defense, antibiotic persistence, and disease pathogenesis . Under stress conditions, VapB17 is likely degraded by cellular proteases, releasing the VapC17 toxin to slow bacterial growth and enabling adaptation to environmental changes. This mechanism is particularly important during infection, where M. tuberculosis must adapt to various host-imposed stresses.

How does VapB17 interact with its cognate toxin VapC17?

VapB17 interacts with VapC17 through direct protein-protein binding, forming a stable complex that neutralizes the ribonuclease activity of VapC17. This interaction typically involves the C-terminal domain of VapB17, which forms an extensive binding interface with VapC17. The binding affinity of this interaction is generally high, ensuring efficient neutralization of the toxin under normal growth conditions. Similar to other studied VapBC systems, the VapB17-VapC17 complex likely also binds to the promoter-operator region of their encoding operon to autoregulate expression .

To experimentally characterize this interaction, researchers should employ multiple complementary approaches:

• Co-expression and co-purification studies using dual expression vectors

• Pull-down assays with tagged versions of either protein

• Surface plasmon resonance (SPR) for quantitative binding kinetics

• Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

• Size-exclusion chromatography to characterize complex formation

What expression systems are most effective for producing functional recombinant VapB17?

Several expression systems can be employed for recombinant VapB17 production, each with distinct advantages:

• E. coli expression systems:

  • pET vectors with T7 promoter offer high yields but may lead to inclusion bodies

  • pBAD vectors with arabinose-inducible promoters provide more controlled expression

  • pCold vectors with cold-shock promoters can improve solubility

• Expression hosts:

  • BL21(DE3) - standard choice for initial trials

  • BL21(DE3)pLysS - tighter control of basal expression

  • Rosetta strains - supply rare codons common in mycobacterial genes

  • SHuffle or Origami strains - enhanced disulfide bond formation if required

• Mycobacterial expression systems:

  • M. smegmatis expression provides a more native-like environment

  • Use of acetamidase promoter for inducible expression

  • Mycobacterial shuttle vectors for propagation in both E. coli and mycobacteria

Co-expression with VapC17 can significantly increase stability and solubility of VapB17, although this approach yields the complex rather than isolated VapB17. Based on studies with other VapB antitoxins, expression at lower temperatures (16-20°C) with reduced inducer concentrations often improves solubility and functional yield .

What are the typical structural characteristics of VapB17 compared to other VapB antitoxins?

VapB17, like other VapB antitoxins, likely possesses a bipartite structural arrangement:

• N-terminal domain:

  • DNA-binding domain, typically containing a ribbon-helix-helix (RHH) or helix-turn-helix (HTH) motif

  • Mediates binding to the operator-promoter region for transcriptional autoregulation

  • Generally more conserved across different VapB proteins

• C-terminal domain:

  • Largely unstructured when not bound to VapC toxin

  • Contains the toxin-neutralizing region that forms a stable interaction interface with VapC17

  • More variable among different VapB proteins, reflecting specificity for cognate toxins

The structure of VapB17 can be characterized using circular dichroism (CD) spectroscopy to assess secondary structure content, NMR spectroscopy for flexible regions, and X-ray crystallography of the VapB17-VapC17 complex. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is valuable for mapping interaction interfaces with both DNA and VapC17 toxin.

What validated antibodies are available for detecting VapB17 in experimental samples?

For detecting VapB17 in experimental samples, researchers should consider these approaches:

• Commercial antibody options:

  • Antibodies against the tag used in recombinant VapB17 (His-tag, FLAG-tag, etc.)

  • Polyclonal antibodies raised against full-length VapB17

  • Custom-made antibodies targeting unique peptide regions of VapB17

• Antibody validation strategies:

  • Use knockout strains (ΔvapB17) as negative controls

  • Include purified recombinant VapB17 as a positive control

  • Perform peptide competition assays to confirm specificity

  • Test antibodies in multiple applications (western blot, immunoprecipitation)

• Alternative detection strategies:

  • Epitope tagging of VapB17 in the native organism at the chromosomal locus

  • Mass spectrometry-based detection and quantification

  • RNA-based detection methods (qRT-PCR) for transcript levels

When selecting antibodies, prioritize those validated for mycobacterial samples or relevant model systems. The approach used for VAPB antibody characterization in search result provides a useful template, where antibodies were systematically evaluated using knockout cell lines and isogenic parental controls to ensure specificity.

How can researchers effectively study cross-interactions between VapB17 and non-cognate VapC toxins?

To systematically investigate potential cross-interactions between VapB17 and non-cognate VapC toxins, researchers should employ a multi-faceted approach:

• In vitro interaction studies:

  • Bacterial two-hybrid or yeast two-hybrid screening against a library of VapC toxins

  • Pull-down assays using tagged VapB17 to capture interacting VapC proteins

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to quantify binding affinities

  • Isothermal titration calorimetry (ITC) for thermodynamic characterization

• Functional neutralization assays:

  • Co-expression of VapB17 with different VapC toxins in E. coli to assess growth rescue

  • In vitro ribonuclease activity assays with purified VapC toxins ± VapB17

  • Structural studies of complexes formed between VapB17 and non-cognate VapCs

Cross-interaction studies are particularly important given the evidence that non-cognate interactions do occur in VapBC systems. For example, research has shown that "VapC35 also interacts with non-cognate antitoxin VapB3" . Such cross-talk significantly impacts the regulatory network complexity and may provide redundancy in stress response pathways. Researchers should also consider that the strength of cross-interactions likely varies considerably, with some non-cognate interactions being significantly weaker than cognate ones but potentially still physiologically relevant.

How does oxidative stress impact VapB17 expression and function in M. tuberculosis?

Based on the behavior of other VapBC systems in M. tuberculosis, the relationship between VapB17 and oxidative stress can be investigated using these approaches:

• Expression analysis under oxidative stress:

  • qRT-PCR to measure vapB17 and vapC17 transcript levels after H₂O₂ or NO exposure

  • Western blotting to track VapB17 protein levels during stress response

  • RNA-seq to place VapB17 regulation in the context of global stress responses

  • Proteomics to detect post-translational modifications of VapB17 during stress

• Functional studies:

  • Construction of vapB17 deletion and overexpression strains

  • Survival assays comparing wild-type and mutant strains under oxidative challenge

  • Complementation studies to confirm phenotype specificity

Research has shown that the "VapBC22 TA system belongs to a key regulatory network and is essential for M. tuberculosis pathogenesis" and "is essential for M. tuberculosis adaptation in oxidative stress conditions" . Notably, "overexpression of VapB22 enhanced the M. tuberculosis susceptibility by ~32-fold and 7-fold upon exposure to oxidative stress for 6 and 24 hours, respectively" . This suggests that precise regulation of VapB17 levels may similarly be critical for oxidative stress adaptation, with either deletion or overexpression potentially compromising bacterial survival under stress conditions.

How can researchers quantitatively assess the impact of VapB17 deletion or overexpression on M. tuberculosis virulence?

For rigorous assessment of VapB17's impact on virulence, researchers should employ multi-level analysis:

• In vitro infection models:

  • THP-1 or primary human macrophage infection assays

  • Quantification of intracellular bacterial survival using CFU counting

  • Flow cytometry-based assessment of macrophage activation markers

  • Cytokine profiling of infected macrophages using ELISA or multiplex assays

• In vivo infection models:

  • Mouse infection models (aerosol, intravenous)

  • Guinea pig model for more human-like pathology

  • Bacterial burden assessment in multiple organs

  • Histopathological analysis of infected tissues

  • Immunological profiling (flow cytometry, cytokine measurements)

• Mechanistic investigations:

  • Transcriptomics of host tissues infected with wild-type vs. mutant strains

  • Proteomics to identify differentially expressed virulence factors

Based on studies of VapBC22, where deletion of vapC22 led to "reduced bacterial loads and lung pathology in guinea pigs" , researchers should expect that manipulation of VapB17 levels might similarly impact virulence. Additionally, transcriptomic analysis revealed that infection with a ΔvapC22 strain resulted in "reduced levels of proinflammatory cytokines in lungs" and increased levels of "anti-inflammatory cytokines, IL-4 and IL-10" . This suggests that comprehensive immunological profiling is essential when assessing the impact of VapB17 on virulence.

What techniques are optimal for studying VapB17's role in transcriptional regulation?

To investigate VapB17's function as a transcriptional regulator, researchers should employ these complementary techniques:

• Identification of binding sites:

  • Electrophoretic mobility shift assays (EMSA) with purified VapB17

  • DNase I footprinting to precisely map protected regions

  • ChIP-seq for genome-wide binding site identification

  • Systematic evolution of ligands by exponential enrichment (SELEX) to identify consensus binding motifs

• Reporter systems:

  • Promoter-reporter fusions (GFP, luciferase) to measure transcriptional activity

  • Dual-reporter systems to normalize for cell number and metabolic state

  • Inducible systems to control VapB17 expression levels

• In vitro transcription assays:

  • Reconstituted transcription systems with purified RNA polymerase

  • Promoter escape assays to study initiation vs. elongation effects

For VapBC35, it has been shown that "an increase in the VapB35 antitoxin to VapC35 toxin ratio results in a stronger binding affinity of the complex with the promoter-operator DNA" . This suggests that for VapB17, the presence and concentration of VapC17 should be carefully controlled in experimental designs, as it likely influences DNA binding activity. Researchers should consider testing VapB17 alone, VapC17 alone, and various ratios of VapB17:VapC17 complex in DNA binding studies to fully characterize the regulatory mechanisms.

What methodological considerations are critical when studying VapB17-mediated stress responses in vitro versus in vivo models?

When comparing VapB17 function across in vitro and in vivo systems, researchers should address these key methodological considerations:

• Stress condition standardization:

  • Define physiologically relevant stress parameters

  • Ensure equivalent stress exposure across models

  • Consider combined stresses that better mimic in vivo conditions

  • Implement gradual stress application rather than sudden exposures

• Temporal dynamics:

  • Capture early vs. late responses through time-course sampling

  • Account for different growth rates in various models

  • Consider stress adaptation vs. acute response distinctions

• Model-specific controls:

  • Include strain background controls relevant to each model

  • Use complementation to confirm phenotype specificity

  • Consider multiple in vivo models with different immune backgrounds

What experimental approaches best characterize VapB17's potential role in antibiotic persistence?

To investigate VapB17's contribution to antibiotic persistence, researchers should implement these systematic approaches:

• Persistence assay design:

  • Time-kill curves with various antibiotic classes

  • Minimum duration for killing (MDK) measurements

  • Persister frequency quantification before and after stress exposure

  • Monitoring regrowth after antibiotic removal

• Genetic manipulation approaches:

  • Phenotypic comparison of ΔvapB17, ΔvapC17, and ΔvapBC17 strains

  • Complementation with wild-type and mutant alleles

  • Inducible overexpression systems to analyze dose-dependent effects

  • Construction of double/triple mutants with other TA systems to address redundancy

• Mechanistic investigations:

  • Transcriptomics of persister-enriched populations

  • Metabolomic profiling to identify persistence-associated metabolic states

  • Single-cell reporters to track gene expression in persisters

TA systems in prokaryotes have been implicated in various biological processes including "antibiotic persistence, and disease pathogenesis" . The mechanism likely involves VapB17 degradation under stress conditions, releasing VapC17 to slow bacterial growth or induce a dormant state that protects against antibiotics targeting actively growing cells. When designing experiments, researchers should consider the potential redundancy among multiple TA systems in M. tuberculosis, as this may mask phenotypes in single-system knockout strains.

How can researchers optimize protocols to study post-translational modifications of VapB17?

To comprehensively characterize post-translational modifications (PTMs) of VapB17, researchers should employ these specialized approaches:

• Mass spectrometry-based identification:

  • Bottom-up proteomics with enrichment for specific modifications

  • Top-down proteomics to analyze intact protein with modifications

  • Targeted methods (MRM/PRM) for quantification of specific modified peptides

  • Phosphoproteomics, acetylomics, or other modification-specific enrichment techniques

• Site-specific characterization:

  • Site-directed mutagenesis of modified residues

  • Expression of phosphomimetic or non-phosphorylatable variants

  • Functional comparison of modified vs. unmodified forms

  • Structural studies to determine modification effects on conformation

• Physiological relevance:

  • Identification of modification enzymes (kinases, acetylases, etc.)

  • Enzyme inhibition or deletion to prevent specific modifications

  • Correlation of modification status with functional outcomes

  • Stress-specific modification patterns

For experimental design, researchers should consider using multiple protein purification methods to preserve labile modifications and include appropriate controls for modification specificity. Based on the behavior of other antitoxins, phosphorylation, acetylation, or proteolytic processing may regulate VapB17 activity, particularly in response to stress conditions that trigger toxin-antitoxin system activation.

How can researchers accurately determine the stoichiometry of VapB17-VapC17 complexes?

For precise determination of VapB17-VapC17 complex stoichiometry, researchers should employ multiple complementary techniques:

• Biophysical approaches:

  • Analytical ultracentrifugation (AUC) to determine molecular mass and shape

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute molecular mass determination

  • Native mass spectrometry to determine complex composition

  • Isothermal titration calorimetry (ITC) to determine binding stoichiometry

• Structural biology methods:

  • X-ray crystallography of the purified complex

  • Cryo-electron microscopy for larger assemblies

  • NMR spectroscopy for solution-state analysis

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Biochemical approaches:

  • Chemical crosslinking followed by mass spectrometry

  • Blue native PAGE for complex integrity assessment

  • Microscale thermophoresis (MST) with labeled components

When analyzing results, researchers should consider that stoichiometry might differ in solution versus crystal structures, concentration-dependent equilibria might exist between different oligomeric states, and the presence of DNA or other binding partners might affect complex formation. Based on other VapBC systems, the VapB17-VapC17 complex likely forms higher-order structures such as heterotetramers (VapB2-VapC2) or more complex arrangements, particularly when bound to DNA.